Contents

OUR TEAM

Name: John Jakoubek

Name: Courtney Willson

Name: Mychal Hooser

Name: Ayanna Akinyemi

Name: Rene Reynolds

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

The images are from Google Images

A Polymerase Chain Reaction (PCR) Machine takes a DNA sequence and makes millions of copies of that sequence. This is done by the experimenter placing the DNA that they desire to copy into the test tube along with DNA polymerase, 2 primers, magnesium chloride, and nucleotides. Once placed in the test tubes and into the machine, the machine has to go through 3 heated steps. The first step is to heat the test tubes to 95 degrees Celsius. This denatures the 2 DNA strands. It is then cooled to 57 degrees Celsius which allows the primers to attach to the appropriate DNA sequences. It is then heated to 72 degrees Celsius. This allows the DNA polymerase to attach to the primers and begin replication. These three steps are repeated 35 times resulting in multiple copies of the desired DNA.

Experimenting With the Connections

When we unplugged the LCD cable from the OpenPCR LCD, the display screen turned off so the readings were no longer available.

When we unplugged the lid temperature sensory wire from the OpenPCR LCD, the machine's temperature of the heating block changed from 26.1 C to -40.0 C.

Test Run

The first test run was on October 23, 2013 at 12:58 p.m. At 1:31 p.m. the machine was showing a temperature of 30.8 degrees Celsius even though it began at 26 degrees Celsius. It was also only on cycle 1. Therefore, it is concluded that this machine is not functioning properly.

Wednesday, October 23, 2013

Protocols

Thermal Cycler Program

DNA Sample Set-up

Positive Control (Patient 1)

Replicate 1

Replicate 2

Replicate 3

Negative Control (Patient 2)

Replicate A

Replicate B

Replicate C

Patient 1 ID: 41731
Patient 2 ID: 78042

DNA Sample Set-up Procedure

Place 50 μL of the PCR reaction mix into each individual tube using a pipette.

Add in 50 μL of the DNA primer mix to the tubes using a clean pipette tip between each tube, being sure not to cross-contaminate the samples.

Place tubes into the PCR machine and close the lid.

PCR Reaction Mix

What is in the PCR reaction mix?

The PCR reaction mix is made up of Taq DNA polymerase, MgCl2 (magnesium chloride), and dNTP's (nucleotides). The polymerase is the main component that functions to replicate the DNA strand. The magnesium chloride is a stabilizer for the polymerase. The dNTP's are the actual nucleotide bases, which make up the DNA strands.

DNA/ primer mix

What is in the DNA/ primer mix?

Each of the DNA primer mixes are made up of different templates of DNA while each mix consists of the same forward primer and reverse primer. These primers are designed to attach to a specific sequence of DNA.

Research and Development

PCR - The Underlying Technology

Component's Functions

A PCR reaction contains five main components: DNA template strand, primers, Taq polymerase, magnesium chloride, and deoxyribonucleotides. The function of the DNA template strand is to do just as the name suggests, act as a template for creating the complementary strand. The primers are pieces of DNA that are specifically designed to match with the desired segment of DNA that is being copied. Being stabilized by the magnesium chloride, Taq plymerase is the component that does the actual replication. Lastly, the deoxyribonucleotides are the actual bases that make up the DNA strands.

Thermal Cycling

In a PCR reaction, three heating steps are taken in order for the reaction to occur. The first step is heating the DNA to 95 degrees Celsius. This denatures the DNA, preparing it for the attachment of the DNA primers. After three minutes, and the DNA is denatured, the reaction is cooled down to 57 degrees Celsius. At this temperature the primers are able to attach to the desired sequence of DNA. This is called annealing. After thirty seconds, the reaction is heated up to 72 degrees Celsius and it expands. During this stage, the polymerase attaches and duplicates the strand. Once the strands are completely duplicated, the reaction is held at 4 degrees Celsius for a short period of time.

Base Pairing

DNA is made up of four nucleotides: adenine, thymine, cytosine, and guanine. Each of these bases can only bind with a specific one of the other bases. Adenine binds with thymine and cytosine with guanine.